Synthesis, intracellular processing and secretion of thrombospondin in human endothelial cells

The biosynthesis of thrombospondin, a glycoprotein first described in platelets, has been studied in human endothelial cells. This glycoprotein has a molecular mass of 450 kDa. It is secreted and incorporated into the extracellular matrix of several cell types in culture. Pulse-chase experiments with [3H]leucine were performed and the synthesis and secretion of the glycoprotein was studied by immunoprecipitation and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The results of these experiments show that the three subunits of thrombospondin are identical in molecular mass. During synthesis there is a small but significant increase in molecular mass within 20 min after pulse labeling. The early form of thrombospondin is sensitive to endoglucosaminidase H treatment, indicating that a transformation of the oligosaccharide structures from 'high-mannose' to 'complex' structures takes place. Within 60 min after synthesis only the mature form of the glycoprotein is secreted into the medium. In the presence of tunicamycin, an inhibitor of N-glycosylation, there is a reduction in molecular mass of the subunit from 165 kDa to 155 kDa. Pulse-chase experiments in the presence of tunicamycin supported the conclusion that the carbohydrate part is processed during biosynthesis. Inhibition of glycosylation had a pronounced effect on the secretion of thrombospondin. The decreased occurrence of thrombospondin in the culture medium seemed to be due to a high intracellular degradation rate of unglycosylated thrombospondin. Characterization of the glycopeptide structures of thrombospondin metabolically labeled with [3H]mannose by Bio-Gel P-4 and concanavalin-A-Sepharose column chromatography revealed that the oligosaccharide structures of the cellular and secreted forms of thrombospondin differ in their composition.     

Complement proteins C2, C4 and factor B. Effect of glycosylation on their secretion and catabolism.

Tunicamycin, an inhibitor of N-acetylglucosaminylpyrophosphopolyisoprenol-dependent glycosylation, was used to study the effect of glycosylation on the synthesis, post-translational modification, secretion and function of the complement proteins that are associated with the major histocompatibility complex in humans, mice and guinea pigs. Tunicamycin blocked glycosylation of pro-C4, C2 and factor B and inhibited secretion of the corresponding native complement proteins synthesized by guinea-pig peritoneal macrophages in tissue culture. In addition, underglycosylated pro-C4 was more rapidly catabolized intracellularly than the corresponding fully glycosylated pro-complement protein. C4 protein secreted by cells incubated with tunicamycin had approximately the same specific biological activity as the protein obtained from control culture media, suggesting that carbohydrate is not required for its activity in immune haemolysis. Direct studies of carbohydrate incorporation and the tunicamycin effect suggested an unequal distribution of sugar among the C4 subunits, with maximal incorporation of carbohydrate into alpha-, and less into the beta-chain of the native protein.     

Effect of N-Linked Glycosylation on Secretion, Activity, and Stability of α-Amylase from Aspergillus oryzae

The effect of N-linked glycosylation on secretion, activity, and stability of α-amylase from Aspergillus oryzae grown as dispersed filaments was studied. In the presence of tunicamycin the fungus grew either as dispersed filaments or as one large pellet, whereas growth was as dispersed filaments in all control cultures. The presence of tunicamycin affected neither biomass, level of secreted α-amylase, nor total amount of secreted protein in cultures growing as dispersed filaments. In these cultures both glycosylated and nonglycosylated α-amylase appeared in the culture medium as well as in the cells, whereas in control cultures only the glycosylated form of α-amylase was found in the medium and in the cells. The presence of nonglycosylated α-amylase in the medium seemed to result from active secretion rather than from autolysis of the mycelium or extracellular deglycosylation. Deglycosylation with Endo H of crude α-amylase in culture filtrate did not affect its stability towards heat, acid pH, or proteolytic degradation. Received: 22 December 1997 / Accepted: 24 February 1998     

Effect of tunicamycin on α-galactosidase secretion by Aspergillus nidulans and the importance of N-glycosylation

Abstract Aspergillus nidulans released α-galactosidase into the culture medium during the exponential growth on either lactose or galactose as the only carbon source. This enzyme is a glycoprotein. Its treatment with endoglycosidases produces a reduction in its molecular mass but the resulting enzyme conserved some of their carbohydrate components in addition to its enzymatic activity. Mycelia of A. nidulans growing in the presence of tunicamycin synthesized an underglycosylated α-galactosidase which was not released into the culture media but remained bound to the cell-wall. Tunicamycin did not prevent the synthesis and secretion of α-galactosidase by protoplasts. N-linked oligosaccharide chains seem not to be essential for the synthesis and secretion of α-galactosidase of A. nidulans , but they could be necessary for proper targeting at the extracellular level.     

Structural analysis of three subunits of laminin from teratocarcinoma-derived parietal endoderm cells

The structure of the three polypeptide chains of the laminin subunits and the number of glycosylation sites in each polypeptide chain were determined using peptide mapping by high-performance liquid chromatography. Analysis of the [³⁵S]methionine-labeled underglycosylated laminin isolated from tunicamycin (TM)-treated cells revealed that the three subunits of laminin contain unique polypeptide chains. Analysis of [³H]glucosamine-labeled glycosylated laminin subunits showed that they are sialylated and that each subunit has 11–14 glycosylation sites.     

Maturation and secretion of lipoprotein lipase in cultured adipose cells. II. Effects of tunicamycin on activation and secretion of the enzyme

The effects of N-linked glycosylation on the activation and secretion of lipoprotein lipase were studied in Ob17 cells. The cells were first depleted of any activity and enzyme content by cycloheximide treatment and of precursors of oligosaccharide chains by tunicamycin. The repletion of lipoprotein lipase content was studied in these cells maintained in the presence of tunicamycin after cycloheximide removal. During the repletion phase, the EC50 values of inhibition by tunicamycin (approx. 0.2 microgram/ml) of the incorporation of labeled glucose, mannose or galactose into trichloroacetic acid-insoluble material were found to be identical. Under these conditions, the rate of protein synthesis was maximally decreased by 30%. The results showed clearly that the recovery in lipoprotein lipase activity was parallel to the recovery in hexose incorporation, no activity being recovered in the absence of glycosylation. An inactive form of lipoprotein lipase from tunicamycin-treated cells was detected by competition experiments with mature active lipoprotein lipase for the binding to immobilized antilipoprotein lipase antibodies, as well as by immunofluorescence staining. SDS-polyacrylamide gel electrophoresis and Western blots of cellular extracts and of extracellular media, obtained after tunicamycin-treated cells were exposed to heparin, revealed a single immunodetectable Mr 52 000 protein, whereas a single Mr 57 000 protein was detected in control cells. Therefore, the results indicate that the acquisition by lipoprotein lipase of a catalytically active conformation is linked directly or indirectly to glycosylation. Despite this lack of activation, the lipoprotein lipase molecule was able to migrate intracellularily and to undergo secretion after heparin stimulation of the tunicamycin-treated cells.     

Functional properties of carbohydrate-depleted tissue plasminogen activator

In order to evaluate the importance of the carbohydrate moiety of human tissue plasminogen activator (TPA), human melanoma (Bowes) cells were treated with a glycosylation inhibitor, tunicamycin (TM), and cellular fractions were assayed for fibrinolytic activity. Where glycosylation was inhibited by 90% and protein synthesis by 30%, TPA specific activity measured by fibrinolytic assays decreased 6-10-fold in the tissue culture medium and cell cytosol with a concomitant 2-fold increase in the 100000g microsomal pellet. In addition, TPA purified to apparent homogeneity was treated with endo-beta-N-acetylglucosaminidase H (Endo-H), producing a fraction that in contrast to native TPA did not adsorb to concanavalin A-Sepharose (Con A-Sepharose). This fraction represented TPA from which 85-90% of N-linked carbohydrate residues had been removed. Native TPA effectively activated plasminogen in the presence of fibrin (Km = 1 microM, kcat = 0.09 s-1) whereas saturation of the enzyme was not achieved at 100 microM plasminogen in the absence of fibrin. Glycosidase-treated and native TPA activated plasminogen at identical high rates in the presence and at identical negligible rates in the absence of fibrin. These studies indicate that the inhibition of glycosylation of TPA results in the inhibition of secretion of the molecule as has been observed for some other glycoproteins. The enzymatic removal of N-linked carbohydrate from purified TPA does not change its unique fibrin-directed properties.     

N-Linked oligosaccharides on the meprin A metalloprotease are important for secretion and enzymatic activity, but not for apical targeting

The alpha and beta subunits of meprins, mammalian zinc metalloendopeptidases, are extensively glycosylated; approximately 25% of the total molecular mass of the subunits is carbohydrate. The aim of this study was to investigate the roles of the N-linked oligosaccharides on the secreted form of mouse meprin A. Recombinant meprin alpha and mutants in which one of the 10 potential Asn glycosylation sites was mutated to Gln were all secreted and sorted exclusively into the apical medium of polarized Madin-Darby canine kidney cells, indicating that no specific N-linked oligosaccharide acts as a determinant for apical targeting of meprin alpha. Several of the mutant proteins had decreased enzymatic activity using a bradykinin analog as substrate, and deglycosylation of the wild-type protein resulted in loss of 75-100% activity. Some of the mutants were also more sensitive to heat inactivation. In studies with agents that inhibit glycosylation processes in vivo, tunicamycin markedly decreased secretion of meprin, whereas castanospermine and swainsonine had little effect on secretion, sorting, or enzymatic properties of meprin. When all the potential glycosylation sites on a truncated form of meprin alpha (alpha-(1-445)) were mutated, the protein was not secreted into the medium, but was retained within the cells even after 10 h. These results indicate that there is no one specific glycosylation site or type of oligosaccharide (high mannose- or complex-type) that determines apical sorting, but that core N-linked carbohydrates are required for optimal enzymatic activity and for secretion of meprin alpha.     

Synthesis and secretion of triacylglycerol lipase by cultured rat hepatocytes

Rat hepatocytes isolated by collagenase perfusion were cultured for 48-72 h and examined for synthesis and secretion of hepatic triacylglycerol lipase activity. Low levels of enzyme activity found in the culture medium increased with time of incubation, and a 3-10-fold rise was encountered in the presence of optimal concentrations of heparin (5 U/ml). After interruption of enzyme synthesis by cycloheximide, plateauing of enzyme activity in the medium occurred, indicating that addition of heparin may not only stabilize but also enhance hepatic triacylglycerol lipase secretion. Synthesis and secretion of hepatic triacylglycerol lipase was not related to cell density, and enzyme secretion was encountered in subconfluent cultures. Release of enzyme activity into the medium was not sensitive to chlorpromazine, a lysosomal enzyme inhibitor, but was completely inhibited by treatment with tunicamycin, an inhibitor of glycosylation. As release of enzyme activity could be maintained for 12 h in the absence of serum, possible hormonal regulation was sought. Under the present experimental conditions, no modulation of hepatic triacylglycerol lipase was encountered by either gonadal or thyroid hormones. Addition of cyclic AMP to the culture medium resulted in a 30% decrease in enzyme activity. The dependence of hepatic triacylglycerol lipase secretion on the intactness of the Golgi apparatus and on vesicular transport was demonstrated by the treatment with monensin. The present results show that cultured rat hepatocytes provide a good model system by which the regulation of synthesis and secretion of hepatic triacylglycerol lipase can be studied.     

Rat hepatocytes in serum-free primary culture elaborate an extensive extracellular matrix containing fibrin and fibronectin

Adult rat hepatocytes cultured on type IV collagen, fibronectin, or laminin and maintained in serum-free medium were examined by indirect immunofluorescence using polyclonal antibodies against extracellular matrix proteins. An extensive fibrillar matrix containing fibronectin and fibrin was detected in all hepatocyte cultures irrespective of the exogenous matrix substratum used to support cell adhesion. Fibrils radiated from the cell periphery and covered the entire culture substratum. In addition, thicker fibers or bundles of fibers were localized on top of hepatocytes. This matrix did not contain laminin or the major types of collagen found in the liver biomatrix (types I, III, and IV). Isolation of the fibrillar matrix and analysis on polyacrylamide gels under reducing conditions demonstrated a major 58-kD polypeptide, derived from beta-fibrinogen as indicated by immunoblotting and two-dimensional peptide mapping. Plasmin rapidly dissolved the matrix. Deposition of the fibrin matrix in hepatocyte cultures was arrested by hirudin, by specific heparin oligosaccharides that potentiate thrombin inhibition by antithrombin III, and by dermatan sulfate, an activator of heparin cofactor II-mediated inhibition of thrombin. The results indicate that hepatocytes in culture synthesize and activate coagulation zymogens. In the absence of inhibitory and fibrinolytic mechanisms, a fibrin clot is formed by the action of thrombin on fibrinogen. Fibronectin attaches to this fibrin clot but fails to elaborate a fibrillar matrix on its own in the presence of coagulation inhibitors.     

Developmental regulation of laminin accumulation in the extracellular matrix of a mouse muscle cell line

We have investigated the synthesis, accumulation, and secretion of laminin, an extracellular matrix glycoprotein, during differentiation of the C2 mouse skeletal muscle cell line in culture. Myoblasts actively synthesized laminin, as measured by incorporation of [35S]methionine and by a dot-immunobinding assay. In myoblast cultures laminin accumulated in an intracellular compartment and could be extracted with a physiological salt solution containing the detergent Triton X-100. After the culture medium was replaced to promote differentiation of myoblasts to myotubes, laminin synthesis was increased, and laminin began to accumulate in the medium in soluble form. During differentiation, laminin also accumulated in an insoluble cell-associated fraction that required guanidinium chloride for extraction. Indirect immunofluorescence and immunobinding assays showed that myotubes but not myoblasts contained laminin on their external surface. The time course of increase in surface laminin paralleled that of the accumulation of insoluble laminin. These results suggest that the insoluble fraction represents laminin bound to the extracellular matrix at the cell surface. Our experiments demonstrate, contrary to previous observations, that myotube cultures synthesize and accumulate laminin, and further, that the differentiation of proliferating myoblasts to multinucleated myotubes is accompanied by increased laminin synthesis, by secretion of laminin into the medium, and by the deposition of laminin into an extracellular matrix on the myotube surface.     

Tunicamycin inhibition of carbohydrate incorporation into thyroglobulin

Carbohydrate chains formation into thyroglobulin (Tg) is a prerequisite for thyroid hormones formation and completeness of carbohydrates chains is necessary for secretion of Tg into the follicles. Tg biosynthesis has been investigated by in vitro experiments, incubating rat thyroid glands with labeled amino-acid and carbohydrate in the presence of tunicamycin, a specific inhibitor of protein glycosylation. Tunicamycin inhibit Tg biosynthesis which is impaired in carbohydrate chains addition but slightly in the polypeptide synthesis, as shown by inhibition of 3H-glucosamine incorporation. Thus tunicamycin inhibits carbohydrate incorporation into Tg without affecting the polypeptide chain growth and decreases its secretion into the follicles.     

The role of the carbohydrate moiety in the biologic properties of fibrinogen

The carbohydrate moiety of some glycoproteins influences their secretion and functional properties. We have examined the importance of the oligosaccharide chains of fibrinogen in this regard. Fibrinogen was labeled de novo by the addition to rabbit hepatocyte monolayer cultures of either 3H-amino-acids or [2-3H] mannose, in the presence or absence of tunicamycin, a potent inhibitor of glycosylation. Inhibition of glycosylation, which ranged from 75 to 80%, was determined by incorporation of [2-3H]mannose as quantitated by gel filtration. Synthesis and secretion of fibrinogen were quantitated by 3H-amino-acid incorporation, using anti-fibrinogen immunoaffinity column chromatography of medium and cell homogenates. Tunicamycin did not appreciably inhibit fibrinogen synthesis, as compared to a 30-40% inhibition of overall protein synthesis, determined by incorporation of 3H-amino-acids into trichloroacetic acid-precipitable material. There was no evidence that secretion of fibrinogen was impaired. Fibrinogen from medium was copurified by adding cold plasma fibrinogen as carrier. Nonglycosylated fibrinogen was found to be functional as demonstrated by incorporation of radioactivity into clots of the copurified material at a rate identical to that of glycosylated fibrinogen. When clotted in the presence of Ca2+ and Factor XIII, cross-linking of glycosylated and nonglycosylated fibrin was demonstrable on fluorography of sodium dodecyl sulfate-polyacrylamide gels, showing disappearance of gamma-chain and appearance of gamma-gamma-dimers.     

Involvement of Glycoproteins in the Development of Early Mouse Embryos: Effect of Tunicamycin and α, α' Dipyridyl in vitro

Early mouse embryos grown in tissue culture were treated with tunicamycin, an inhibitor of protein glycosylation or with αα' dipyridyl, an inhibitor of collagen secretion. Neither treatment blocked development of cleavage stage embryos nor did either interfere with blastocyst formation, hatching, or adhesion to the substratum at low concentrations. However, both treatments caused marked and specific changes in the morphology of the blastocyst outgrowth. Treatment of embryos with tunicamycin caused severe deterioration of the trophoblast layer and subsequent disintegration of the inner cell mass. Tunicamycin completely inhibited the incorporation of mannose into proteins. Treatment with αα' dipyridyl caused dose dependent retardation of the inner cell mass while the trophoblast cells were virtually unaffected. These alterations in morphogenesis occurred only in embryos treated at the blastocyst stage or later in development. Changes caused by α,α' dipyridyl could be partially reversed by addition of collagen to the culture. These findings might indicate the involvement of extracellular matrix macromolecules in embryonic organization.     

Marmoset monkey trophoblastic tissue growth and matrix metalloproteinase secretion in culture

Marmoset monkey blastocysts maintained in culture produced trophoblastic vesicles up to 4 mm in diameter that were subdivided into fragments and subcultured to produce new vesicles. These tissues are composed of an outer layer of trophoblast-like cells and an inner layer of endoderm-like cells, and resemble a blastocyst wall. When such vesicles were cultured in serum-free medium for 14 days, they increased in size but there was no significant difference in their protein content at the end of culture. The proliferation index, measured by BrdU incorporation, varied considerably within and between vesicles. The purpose of this investigation was to determine which matrix metalloproteinases are secreted by marmoset monkey trophoblastic tissue in vitro, and the effect of extracellular laminin on this secretion. It was determined by zymography that the vesicles secreted matrix metalloproteinase 2, but not matrix metalloproteinase 9, and that matrix metalloproteinase 2 was secreted as the proenzyme (72 kDa). Matrix metalloproteinases 1, 3 and 7 were not detectable in the culture medium. The addition of laminin (5-20 micrograms ml-1), either as a substrate or in solution in the medium, did not have a consistent effect on matrix metalloproteinase 2 secretion during the culture period. The vesicles were found to express both matrix metalloproteinases 2 and 9 in both types of cell when examined by immunohistochemistry. The expression of matrix metalloproteinase 9 in the vesicles, but the absence of its secretion, indicates that specific factors, possibly of endometrial origin, may be required for inducing secretion.     

Responses of cultured neural retinal cells to substratum-bound laminin and other extracellular matrix molecules

The responses of cultured chick embryo retinal neurons to several extracellular matrix molecules are described. Retinal cell suspensions in serum-free medium containing the "N1" supplement (J. E. Bottenstein, S. D. Skaper, S. Varon, and J. Sato, 1980, Exp. Cell Res. 125, 183-190) were seeded on tissue culture plastic surfaces pretreated with polyornithine (PORN) and with one of the factors to be tested. Substantial cell survival could be observed after 72 hr in vitro on PORN pretreated with serum or laminin, whereas most cells appeared to be degenerating on untreated PORN, PORN-fibronectin, and PORN-chondronectin. Cell attachment, although quantitatively similar for all these substrata, was temperature-dependent on serum and laminin but not on fibronectin or untreated PORN. In a short-term bioassay, neurite development was abundant on laminin, scarce on serum and fibronectin, and absent on PORN. No positive correlation between cell spreading and neurite production could be seen: cell spreading was more extensive on PORN and fibronectin than on laminin or serum, while on laminin-treated dishes, spreading was similar for neurite-bearing and non-neurite-bearing cells. Laminin effects on retinal neurons were clearly substratum dependent. When bound to tissue culture plastic, laminin showed a dose-dependent inhibitory effect on cell attachment and did not stimulate neurite development. PORN-bound laminin, on the other hand, did not affect cell attachment but caused marked stimulation of neurite development, suggesting that laminin conformation and/or the spatial distribution of active sites play an important role in the neurite-promoting function of this extracellular matrix molecule. Investigation of the embryonic retina with ELISA and immunocytochemical methods showed that laminin is present in this organ during development. Therefore, in vivo and in vitro observations are consistent with the possibility that laminin might influence neuronal development in the retina.     

Abnormal laminin secretion from parietal endoderm-like F9 cells in the presence of monensin

We studied the effects of monensin on post-translational modification and intracellular transport of precursors of laminin subunits in parietal endoderm-like F9 cells. At concentrations higher than 0.1 microM, monensin inhibited the processing of high-mannose type precursors for all three subunits and caused their cytoplasmic accumulation. Furthermore, the secretion of mature subunits of laminin was inhibited. Instead, polypeptides with similar molecular weights to those of intracellular precursors were secreted. These polypeptides were immunologically related to laminin subunits and were sensitive to digestion with beta-N-acetylglucosaminidase H (Endo H). This indicated that Golgi complexes of the cells can transport the precursors of laminin subunits even with their terminal glycosylation inactivated by monensin. Tunicamycin induced the accumulation of unglycosylated precursors and strongly reduced their secretion into the medium.     

Palmitylation, sulfation, and glycosylation of the alpha subunit of the sodium channel. Role of post-translational modifications in channel assembly

Antibodies to the alpha and beta 2 subunits and site-directed antibodies that distinguish alpha subunits of the RI and RII subtypes have been used to study the biosynthesis and assembly of sodium channels. The RII sodium channel subtype is preferentially expressed in rat brain neurons in primary cell culture. Post-translational processing of alpha subunits includes incorporation of palmityl residues in thioester linkage and sulfate residues attached to oligosaccharides. The incorporation of [3H] palmitate into alpha subunits is inhibited by tunicamycin, indicating that it occurs in the early stages of biosynthesis but after co-translational glycosylation. Mature alpha subunits are attached to beta 2 subunits through disulfide bonds within 1 h after synthesis and up to 30% can be specifically immunoprecipitated from the cell surface with antibodies against the beta 2 subunits by 4 h after synthesis. The remaining alpha subunits remain in an intracellular pool. The alpha subunits synthesized in the presence of castanospermine and swainsonine have reduced apparent size. Castanospermine prevents incorporation of approximately 81% of the sialic acid of the alpha subunit and inhibits sulfation but not palmitylation. Although inhibition of glycosylation with tunicamycin blocks assembly of functional sodium channels, castanospermine and swainsonine do not prevent the covalent assembly of alpha and beta 2 subunits or the transport of alpha beta 2 complexes to the cell surface, and sodium channels synthesized under these conditions have normal affinity for saxitoxin. Thus, the extensive processing and terminal sialylation of oligosaccharide chains during maturation of the alpha subunit is not essential. A kinetic model for biosynthesis, processing, and assembly of sodium channel subunits is presented.     

Effect of tunicamycin on the secretion of serum proteins by primary cultures of rat and chick hepatocytes. Studies on transferrin, very low density lipoprotein, and serum albumin.

Using rat or chick hepatocyte monolayers, we have studied the effect of tunicamycin, a specific inhibitor of protein glycosylation, on the synthesis and secretion of serum proteins. Tunicamycin inhibited glucosamine incorporation into rat liver transferrin and the apoprotein B chain of chick liver very low density lipoprotein (VLDL) by 75 to 90%. In contrasts, amino acid incorporation into these two glycoproteins, as well as into the normally unglycosylated proteins, rat serum albumin and apoprotein A of chick liver VLDL, was decreased by only 10 to 25% in the presence of the antibiotic. Despite the inhibitory effect of tunicamycin on glycosylation, secretion of all four proteins was virtually unimpaired. Thus, the carbohydrate moieties of rat liver transferrin or apoprotein B of chick liver VLDL do not appear to play an essential role in the secretion process.     

Tunicamycin does not block ovalbumin secretion in the oviduct

In order to examine the role of carbohydrate in the secretion of ovalbumin, oviduct minces were incubated in the presence of tunicamycin, an inhibitor of dolichol-mediated glycosylation. Ovalbumin secretion was monitored immunologically and found to be identical, within experimental error, in the absence and presence of tunicamycin. These results, coupled with the recent finding of Palmiter $\text{et}\text{al}$. [Proc. Natl. Acad. Sci. (1978) $\text{75}$, 94–98] indicate that neither a transient hydrophobic pre-piece nor carbohydrate is required for ovalbumin secretion.