1-deoxynojirimycin impairs oligosaccharide processing of alpha 1-proteinase inhibitor and inhibits its secretion in primary cultures of rat hepatocytes

1-Deoxynojirimycin was found to inhibit oligosaccharide processing of rat alpha 1-proteinase inhibitor. In normal hepatocytes alpha 1-proteinase inhibitor was present in the cells as a 49,000 Mr high mannose type glycoprotein with oligosaccharide side chains having the composition Man9GlcNAc and Man8GlcNAc with the former in a higher proportion. Hepatocytes treated with 5 mM 1-deoxynojirimycin accumulated alpha 1-proteinase inhibitor as a 51,000 Mr glycoprotein with carbohydrate side chains of the high mannose type, containing glucose as measured by their sensitivity against alpha-glucosidase, the largest species being Glc3Man9GlcNAc. Conversion to complex oligosaccharides was inhibited by the drug. In addition, increasing concentrations of 1-deoxynojirimycin inhibited glycosylation resulting in the formation of some alpha 1-proteinase inhibitor with two instead of three oligosaccharide side chains. 5 mM 1-deoxynojirimycin inhibited the secretion of alpha 1-proteinase inhibitor by about 50%, whereas secretion of albumin was unaffected. The oligosaccharides of alpha 1-proteinase inhibitor secreted from 1-deoxynojirimycin-treated cells were characterized by their susceptibility to endoglucosaminidase H, incorporation of [3H]galactose, and [3H]fucose and concanavalin A-Sepharose chromatography. It was found that 1-deoxynojirimycin did not completely block oligosaccharide processing, resulting in the formation of alpha 1-proteinase inhibitor molecules carrying one or two complex type oligosaccharides. Only these alpha 1-proteinase inhibitor molecules processed to the complex type in one or two of their oligosaccharide chains were nearly exclusively secreted. This finding demonstrates the importance of oligosaccharide processing for the secretion of alpha 1-proteinase inhibitor.     

Effect of swainsonine on the processing of the asparagine-linked carbohydrate chains of alpha 1-antitrypsin in rat hepatocytes. Evidence for the formation of hybrid oligosaccharides

The biosynthesis of the proteinase inhibitor alpha 1-antitrypsin has been studied in rat hepatocyte primary cultures. Newly synthesized alpha 1-antitrypsin was found in hepatocytes as a glycoprotein of an apparent molecular weight of 49,000 carrying oligosaccharide side chains of the high mannose type. In the hepatocyte medium a secreted alpha 1-antitrypsin of an apparent molecular weight of 54,000 could be identified as a glycoprotein with carbohydrate chains of the complex type. Pulse-chase experiments revealed a precursor-product relationship for the two forms of alpha 1-antitrypsin. When the hepatocytes were treated with swainsonine, an intracellular form of alpha 1-antitrypsin with an apparent molecular weight of 49,000 indistinguishable from that of control cells was found. However, the alpha 1-antitrypsin secreted from swainsonine-treated hepatocytes was different from that present in control media. It was characterized by a lower apparent molecular weight (51,000), a higher amount of [3H]mannose incorporation, half as much incorporation of [3H]galactose, and the same amount of [3H]fucose incorporation compared to alpha 1-antitrypsin of control media. In contrast to the 54,000 complex type alpha 1-antitrypsin from control media the 51,000 alpha 1-antitrypsin from the medium of swainsonine-treated cells was found to be susceptible to the action of endoglucosaminidase H, even when fucose was attached to the proximal GlcNAc residue. alpha 1-Antitrypsin secreted from swainsonine-treated cells combines features usually associated with either high mannose or complex type oligosaccharides and therefore represents a hybrid structure. In spite of its effect on the carbohydrate part of alpha 1-antitrypsin swainsonine did not impair the secretion of the incompletely processed glycoprotein.     

Different effects of the glucosidase inhibitors 1-deoxynojirimycin, N-methyl-1-deoxynojirimycin and castanospermine on the glycosylation of rat alpha 1-proteinase inhibitor and alpha 1-acid glycoprotein.

The glucosidase inhibitors 1-deoxynojirimycin, N-methyl-1-deoxynojirimycin and castanospermine were used to inhibit oligosaccharide processing in primary cultures of rat hepatocytes. Their effect on the glycosylation of alpha 1-proteinase inhibitor (alpha 1PI) and alpha 1-acid glycoprotein (alpha 1AGP) was studied. Of the three glucosidase inhibitors examined, 1-deoxynojirimycin inhibited not only oligosaccharide trimming but also glycosylation de novo of newly synthesized proteins, resulting in the formation of alpha 1PI with two and three (normally carrying three) and alpha 1AGP with two to five (normally carrying six) oligosaccharide side chains. In the presence of the glucosidase inhibitors, glucosylated high-mannose-type oligosaccharides accumulated. Whereas most of the endoglucosaminidase-H-sensitive oligosaccharides formed in the presence of 1-deoxynojirimycin contained only one glucose residue, N-methyl-1-deoxynojirimycin and castanospermine led mainly to the formation of oligosaccharides with three glucose residues. None of the three glucosidase inhibitors completely prevented the formation of complex-type oligosaccharides. Thus, in their presence, alpha 1PI and alpha 1AGP with a mixture of both high-mannose and complex-type oligosaccharides were secreted.     

Effect of the threonine analog beta-hydroxynorvaline on the glycosylation and secretion of alpha 1-acid glycoprotein by rat hepatocytes

The threonine analog beta-hydroxynorvaline (Hnv) is an inhibitor of asparagine-linked glycosylation. In the presence of the analog hepatocytes synthesized immunoreactive alpha 1-acid glycoprotein with 0-6 oligosaccharide chains. Pulse-chase experiments were conducted to compare the rates of secretion of alpha 1-acid glycoprotein from untreated, tunicamycin-treated, and Hnv-treated cells. Partially glycosylated (1-5 oligosaccharide chains) and unglycosylated (tunicamycin-inhibited) molecules exited the cells more slowly than native alpha 1-acid glycoprotein. In addition, secretion of fully glycosylated (6 oligosaccharide chains) alpha 1-acid glycoprotein was retarded in Hnv-treated cells when compared to controls. The slowest rate of secretion was exhibited by the unglycosylated form from Hnv-treated cells. These results suggest that Hnv-induced changes either in the extent of glycosylation or in the peptide sequence of alpha 1-acid glycoprotein can interfere with its transport through the cell. The major intracellular forms of alpha 1-acid glycoprotein from control and Hnv-treated cells were endoglycosidase H-sensitive and contained Man9-8 GlcNAc2 oligosaccharide structures. The oligosaccharide chains on the secreted molecules from control and Hnv-treated cells were entirely of the endoglycosidase H-resistant, complex type.     

Biosynthesis and secretion of M- and Z-type alpha 1-proteinase inhibitor by human monocytes. Effect of inhibitors of glycosylation and of oligosaccharide processing on secretion and function

The biosynthesis and secretion of M-type and Z-type alpha 1-antitrypsin was studied in human monocytes. In monocytes of PiMM individuals alpha 1-antitrypsin represented 0.08% of the newly synthesized proteins and 0.44% of the secreted proteins. Two molecular forms of alpha 1-antitrypsin could be identified: a 51-kDa intracellular form, susceptible to endoglucosaminidase H, thus representing the high-mannose type precursor form and a 56-kDa form resistant to endoglucosaminidase H which was secreted into the medium. Inhibition of de novo glycosylation by tunicamycin impaired the secretion of M-type alpha 1-antitrypsin by about 75% whereas inhibition of oligosaccharide processing by the mannosidase II inhibitor swainsonine did not alter the secretion of M-type alpha 1-antitrypsin. alpha 1-Antitrypsin secreted by human monocytes was functionally active as measured by complex formation with porcine pancreatic elastase. Even unglycosylated alpha 1-antitrypsin secreted by human monocytes treated with tunicamycin formed a complex with elastase. In monocytes of PiZZ individuals the secretion of alpha 1-antitrypsin was decreased. 72% of newly synthesized M-type alpha 1-antitrypsin, but only 35% of newly synthesized Z-type alpha 1-antitrypsin were secreted during a labeling period of 3 h with [35S]methionine. The 51-kDa form of Z-type alpha 1-antitrypsin accumulated intracellularly, whereas the 56-kDa form was secreted. Inhibition of oligosaccharide processing by swainsonine did not alter the decreased secretion of Z-type alpha 1-antitrypsin, whereas inhibition of de novo glycosylation by tunicamycin blocked the secretion of Z-type alpha 1-antitrypsin completely.     

Disparate effects of monensin and colchicine on intracellular processing of secretory proteins in cultured rat hepatocytes

We have studied the biosynthesis and intracellular processing of three major secretory proteins, albumin, alpha 1-protease inhibitor and alpha 2u-globulin, in cultured rat hepatocytes. The effect of secretion-blocking agents, monensin, a monovalent ionophore, and the microtubule-affecting agents colchicine and taxol was determined. In the control cells, alpha 1-protease inhibitor, a glycoprotein, was first synthesized as an endoglycosidase-H-sensitive form with Mr 51 000, and then processed to two endoglycosidase-H-resistant forms having Mr 51 000 and 56 000, the latter of which was secreted into the medium. Initially synthesized proalbumin was converted with chase to serum-type albumin, while no pro-type precursor was identified for alpha 2u-globulin. In the cells treated with colchicine or taxol, in which secretion was greatly inhibited, the fully processed alpha 1-protease inhibitor and albumin accumulated and were finally secreted into the medium. In the monensin-treated cells, however, most of the newly synthesized alpha 1-protease inhibitor and albumin were not processed to the final mature forms, resulting in accumulation of two 51 000-Mr forms and proalbumin, respectively. Moreover in treated cells, proalbumin and the endoglycosidase-H-resistant alpha 1-protease inhibitor were finally secreted into the medium. Such an effect was not caused by NH4Cl which also inhibited the secretion and is known to exert the similar effect as monensin on the receptor-mediated endocytosis pathway. Based on these results, the use of monensin may prove valuable for more detailed analysis of intracellular processing of various proteins.     

Involvement of various organs in the initial plasma clearance of differently glycosylated rat liver secretory proteins

The initial plasma clearance and organ distribution of alpha 1-acid glycoprotein and alpha 2-macroglobulin carrying different types of oligosaccharide, side chains was studied in rats. The differently glycosylated proteins were synthesized by rat hepatocytes in culture in the presence of tunicamycin (unglycosylated form), swainsonine (hybrid type), or 1-deoxymannojirimycin (high-mannose type). Deglycosylated glycoproteins (Asn-GlcNAc) were obtained by endoglucosaminidase H treatment of high-mannose-type glycoproteins. Ten minutes after intravenous injection 3% of complex type, 26% of hybrid type, 84% of high-mannose type. 64% of unglycosylated and 80% of deglycosylated alpha 1-acid glycoprotein disappeared from the plasma. The respective values for alpha 2-macroglobulin were 26%, 42%, 59% and 67%. When the clearance of total hepatic secretory proteins was examined, major differences between glycosylated and unglycosylated (glyco)proteins were found, particularly in the case of low-molecular-mass polypeptides. Whereas complex-type alpha 1-acid glycoprotein and alpha 2-macroglobulin showed no accumulation in various organs, hybrid-type alpha 1-acid glycoprotein and alpha 2-macroglobulin were present in spleen and liver. High-mannose-type alpha 1-acid glycoprotein and alpha 2-macroglobulin also accumulated mainly in spleen and liver. Spleen had the highest specific activity; liver, due to its larger organ mass, represented the major organ for the uptake of high-mannose-type glycoproteins. Competition experiments with mannan and GlcNAc-bovine-serum-albumin showed a mannose/GlcNAc receptor-mediated removal. Whereas unglycosylated alpha 1-acid glycoprotein was taken up by the kidney, unglycosylated alpha 2-macroglobulin was found in the spleen. Deglycosylated glycoproteins (Asn-GlcNAc) were removed from the plasma via two different mechanisms: firstly, clearance by the kidney similar to the unglycosylated glycoproteins; secondly, clearance by a mannose/GlcNAc receptor-mediated uptake mainly into the spleen. We conclude that N-linked oligosaccharide side chains are important for the plasma survival of hepatic secretory glycoproteins and that unphysiologically glycosylated forms are cleared by different mechanisms.     

Unglycosylated rat alpha 1-proteinase inhibitor has a six-fold shorter plasma half-life than the mature glycoprotein

The plasma half-lives of glycosylated and unglycosylated alpha 1-proteinase inhibitor-radioactively labeled with [35S]methionine in rat hepatocyte primary cultures - were determined in the rat. Unglycosylated alpha 1-proteinase inhibitor was synthesized by hepatocytes in the presence of tunicamycin. Media from hepatocytes containing 35S-labeled glycosylated or unglycosylated alpha 1-proteinase inhibitor were injected into the tail veins of rats. At different times after injection alpha 1-proteinase inhibitor was isolated from plasma by affinity chromatography with anti-alpha 1-proteinase inhibitor Sepharose. Radioactivity measurements revealed a plasma half-life of 170 min for glycosylated alpha 1-proteinase inhibitor and of 30 min for the unglycosylated form of the inhibitor.     

The role of N-glycosylation for the plasma clearance of rat liver secretory glycoproteins

The clearance of total rat liver secretory glycoproteins and of alpha 1-acid glycoprotein carrying no or different types of oligosaccharide side chains was studied in vivo and in the isolated perfused rat liver. In order to obtain unglycosylated or differently glycosylated forms of secreted glycoproteins, rat hepatocyte primary cultures were incubated with various inhibitors of N-glycosylation. Tunicamycin was used for the synthesis of unglycosylated (glyco)proteins, the mannosidase I inhibitor 1-deoxymannojirimycin for the synthesis of high-mannose type and the mannosidase II inhibitor swainsonine for the synthesis of hybrid-type glycoproteins. Glycoproteins carrying carbohydrate side chains of the complex type were synthesized by control hepatocytes. In vivo and in the perfused rat liver, high-mannose-type glycoproteins were cleared at the highest rate, followed by unglycosylated and hybrid-type glycoproteins. The lowest clearance rate was found for the glycoproteins with carbohydrate side chains of the complex type. For the highly glycosylated alpha 1-acid glycoprotein the differences in clearance rates were more pronounced. The following plasma half-lives were determined in vivo: complex type, 100 min; hybrid type, 15 min; unglycosylated form, 5 min; and high-mannose type less than 1 min. In the recirculating perfused liver 28% of complex-type alpha 1-acid glycoprotein, 40% of hybrid type, 47% of unglycosylated and 93% of high-mannose-type alpha 1-acid glycoprotein were removed from the perfusate within 2 h. It is concluded that N-glycosylation and processing to complex-type oligosaccharides seems to be of great importance for the circulatory life time of plasma glycoproteins.     

Hepatocyte specific long lasting inhibition of protein N-glycosylation by D-galactosamine

The effect of D-galactosamine on protein N-glycosylation was studied in rat hepatocyte primary cultures for alpha 1-antitrypsin (three complex type oligosaccharide chains) and alpha 1-acid glycoprotein (six complex type oligosaccharide chains). D-Galactosamine at a concentration of 4 mM inhibited partially de novo N-glycosylation leading to the formation of alpha 1-antitrypsin lacking one to two and of alpha 1-acid glycoprotein lacking one to five of its carbohydrate side chains. In addition D-galactosamine interfered with oligosaccharide processing, leading to the formation of some carbohydrate side chains remaining in an endoglucosaminidase H sensitive, i.e., not completely processed, form. D-Galactosamine impaired the secretion of alpha 1-antitrypsin and of alpha 1-acid glycoprotein but did not inhibit the secretion of the unglycosylated albumin. The inhibitory effect of D-galactosamine on de novo glycosylation as well as on oligosaccharide processing lasted for at least 24 h after it had been removed from the cells. D-Galactosamine impaired the glycosylation of alpha 1-antitrypsin only in hepatocytes, but not in human monocytes. Furthermore, D-galactosamine did not impair the N- and O-glycosylation of interleukin-6 in human monocytes and in MRC 5 fibroblasts. The results indicate that the effect of D-galactosamine on protein glycosylation is restricted to D-galactosamine metabolizing hepatocytes and is not exerted by the drug itself but by its metabolites.     

Non-carrier-mediated uptake of the mannosidase I inhibitor 1-deoxymannojirimycin by K562 erythroleukemic cells

A 3H label was introduced at the C-1 position of the mannosidase I inhibitor 1-deoxymannojirimycin (dMM) by catalytic hydrogenolysis of benzyl-2,3-O-isopropylidene-5-N-benzyl-6-O-benzyl-alpha-D-mannofurano side with 3H2. 1-[3H]dMM as well as its precursor 1-[3H]2,3-O-isopropylidene-dMM had identical Rf as the nonradioactive compounds on TLC. Furthermore, alpha 1-antitrypsin secreted by HepG2 cells was modified indistinguishably by treatment of the cells with dMM and 1-[3H]dMM. Thus, 1-[3H]dMM had chemical and biological properties identical with authentic dMM. Uptake of [14C]mannose by K562 cells could be inhibited by glucose but not by the mannose analogue dMM. Thus, dMM does not enter the cell through hexose transporter(s). Uptake of 1-[3H]dMM by K562 cells could not be inhibited by increasing concentrations of nonradioactive dMM (from 1-32,000 microM), showing transport of dMM into cells through nonfacilitated diffusion. Furthermore, uptake of 1-[3H]dMM by K562 cells was observed at 0 degrees C.     

Qualitative studies of lung lavage alpha 1-proteinase inhibitor

A method is described which enables identification of the molecular size of alpha 1-proteinase inhibitor (alpha 1-PI) in biological fluids. This technique when applied to bronchoalveolar lavage fluids clearly demonstrates alpha 1-PI in three molecular forms; the native molecule (Mr approximately equal to ++54 000), a partially proteolysed form (Mr approximately equal to 49 000) and in a form suggestive of a complex with enzyme (Mr approximately equal to 82 000). Samples showing the presence of native alpha 1-PI inhibited more porcine pancreatic elastase than samples where no native alpha 1-PI was seen or where the predominant form was partially proteolysed alpha 1-PI (p less than 0.01). Although the predominant band of alpha 1-PI was more frequently the partially proteolysed form in current smokers (p less than 0.01), there was no clear difference in the inhibitory function of alpha 1-PI between current smokers and non-smokers and those with and without airflow obstruction.     

Clearance of acute-phase plasma proteins with no, high-mannose-, hybrid-, or complex type oligosaccharide side chains by the isolated perfused rat liver

The clearance of the rat acute-phase proteins alpha 2-macroglobulin, alpha 1-proteinase inhibitor and alpha 1-acid glycoprotein with no, high-mannose, hybrid or complex type oligosaccharide side chains was determined in the isolated perfused rat liver. The differently glycosylated forms of the three proteins were obtained from rat hepatocyte primary cultures treated with different inhibitors of glycosylation. The complex type forms of the three proteins were essentially not cleared by the liver during 2 h of perfusion. Unglycosylated alpha 2-macroglobulin and alpha 1-acid glycoprotein decreased in the perfusate by about 50% after 2 h; unglycosylated alpha 1-proteinase inhibitor was not taken up by the liver. The high-mannose type forms of the three proteins were nearly totally cleared. After 2 h of perfusion 10%, 45% and 30% of the hybrid type forms of alpha 2-macroglobulin, alpha 1-proteinase inhibitor and alpha 1-acid glycoprotein, respectively, were cleared. The clearance rates of high-mannose and of hybrid type glycoproteins could be reduced to the rates of complex type glycoproteins by the addition of mannan to the perfusate. It is concluded that complex type glycosylation prevents the uptake of plasma glycoproteins by the liver.     

Hepatocyte uptake of alpha 1-proteinase inhibitor-trypsin complexes in vitro: evidence for a shared uptake mechanism for proteinase complexes of alpha 1-proteinase inhibitor and antithrombin III

In vivo clearance studies have indicated that the clearance of proteinase complexes of the homologous serine proteinase inhibitors alpha 1-proteinase inhibitor and antithrombin III occurs via a specific and saturable pathway located on hepatocytes. In vitro hepatocyte-uptake studies with antithrombin III-proteinase complexes confirmed the hepatocyte uptake and degradation of these complexes, and demonstrated the formation of a disulfide interchange product between the ligand and a cellular protein. We now report the results of in vitro hepatocyte uptake studies with alpha 1-proteinase inhibitor-trypsin complexes. Trypsin complexes of alpha 1-proteinase inhibitor were prepared and purified to homogeneity. Uptake of these complexes by hepatocytes was time and concentration-dependent. Competition experiments with alpha 1-proteinase inhibitor, alpha 1-proteinase inhibitor-trypsin, and antithrombin III-thrombin indicated that the proteinase complexes of these two inhibitors are recognized by the same uptake mechanism, whereas the native inhibitor is not. Uptake studies were performed at 37 degrees C with 125I-alpha 1-proteinase inhibitor-trypsin and analyzed by sodium dodecyl sulfate-gel electrophoresis in conjunction with autoradiography. These studies demonstrated time-dependent uptake and degradation of the ligand to low molecular weight peptides. In addition, there was a time-dependent accumulation of a high molecular weight complex of ligand and a cellular protein. This complex disappeared when gels were performed under reducing conditions. The sole cysteine residue in alpha 1-proteinase inhibitor was reduced and alkylated with iodoacetamide. Trypsin complexes of the modified inhibitor were prepared and purified to homogeneity. Uptake and degradation studies demonstrated no differences in the results obtained with this modified complex as compared to unmodified alpha 1-proteinase inhibitor-trypsin complex. In addition, the high molecular weight disulfide interchange product was still present on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized cells. Clearance and clearance competition studies with alpha 1-proteinase inhibitor-trypsin, alkylated alpha 1-proteinase inhibitor-trypsin, antithrombin III-thrombin, and anti-thrombin III-factor IXa further demonstrated the shared hepatocyte uptake mechanism for all these complexes.     

Biosynthesis and secretion of alpha 1 acute-phase globulin in primary cultures of rat hepatocytes

Experimental inflammation in rats led to a sevenfold increase in serum levels of alpha 1 acute-phase globulin. This increase is correlated with elevated levels of translatable mRNA for alpha 1 acute-phase globulin in the liver. Biosynthesis and secretion of alpha 1 acute-phase globulin were studied in rat hepatocyte primary cultures. An intracellular form of alpha 1 acute-phase globulin with an apparent relative molecular mass of 63 500 and a secreted form of 68 000 were found. The intracellular form of alpha 1 acute-phase globulin could be deglycosylated by endoglucosaminidase H treatment indicating that its oligosaccharide chains were of the high-mannose type. The secreted form of alpha 1 acute-phase globulin was not sensitive to endoglucosaminidase H, but was susceptible to the action of sialidase reflecting carbohydrate side-chains of the complex type. Pulse-chase experiments revealed a precursor-product relationship for the high-mannose and the complex type alpha 1 acute-phase globulin. In the hepatocyte medium newly synthesized alpha 1 acute-phase globulin was detected 30 min after the pulse. Unglycosylated alpha 1 acute-phase globulin was found in the cells as well as in the medium when the transfer of oligosaccharide chains onto the polypeptide chains was blocked by tunicamycin. Tunicamycin led to a marked delay in alpha 1 acute-phase globulin secretion.     

Acute phase protein alpha 1-acid glycoprotein interacts with plasminogen activator inhibitor type 1 and stabilizes its inhibitory activity.

alpha(1)-Acid glycoprotein, one of the major acute phase proteins, was found to interact with plasminogen activator inhibitor type 1 (PAI-1) and to stabilize its inhibitory activity toward plasminogen activators. This conclusion is based on the following observations: (a) alpha(1)-acid glycoprotein was identified to bind PAI-1 by a yeast two-hybrid system. Three of 10 positive clones identified by this method to interact with PAI-1 contained almost the entire sequence of alpha(1)-acid glycoprotein; (b) this protein formed complexes with PAI-1 that could be immunoprecipitated from both the incubation mixtures and blood plasma by specific antibodies to either PAI-1 or alpha(1)-acid glycoprotein. Such complexes could be also detected by a solid phase binding assay; and (c) the real-time bimolecular interactions monitored by surface plasmon resonance indicated that the complex of alpha(1)-acid glycoprotein with PAI-1 is less stable than that formed by vitronectin with PAI-1, but in both cases, the apparent K(D) values were in the range of strong interactions (4.51 + 1.33 and 0.58 + 0.07 nm, respectively). The on rate for binding of PAI-1 to alpha(1)-glycoprotein or vitronectin differed by 2-fold, indicating much faster complex formation by vitronectin than by alpha(1)-acid glycoprotein. On the other hand, dissociation of PAI-1 bound to vitronectin was much slower than that from the alpha(1)-acid glycoprotein, as indicated by 4-fold lower k(off) values. Furthermore, the PAI-1 activity toward urokinase-type plasminogen activator and tissue-type plasminogen activator was significantly prolonged in the presence of alpha(1)-acid glycoprotein. These observations suggest that the complex of PAI-1 with alpha(1)-acid glycoprotein can play a role as an alternative reservoir of the physiologically active form of the inhibitor, particularly during inflammation or other acute phase reactions.     

Deglycosylation of alpha 1-proteinase inhibitor by endo-beta-N-acetylglucosaminidase F

The glycosidase endo-beta-N-acetylglucosaminidase F (endo F) from Flavobacterium meningosepticum was used for the deglycosylation of rat alpha 1-proteinase inhibitor (alpha 1 PI). alpha 1 PI containing three oligosaccharide side chains of the complex type was isolated from rat serum or from the medium of rat hepatocyte primary cultures. High-mannose-type alpha 1 PI or hybrid-type alpha 1 PI was isolated from the media of hepatocytes treated with 1-deoxymannojirimycin or swainsonine, respectively. The susceptibility of complex-type alpha 1 PI to endo F was studied in the presence of various detergents. 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate and octyl glucopyranoside turned out to be most effective. In the absence of detergents, digestion of alpha 1 PI with high concentrations of endo F and/or long times of incubation led to the formation of alpha 1 PI with one and two oligosaccharide side chains. In the presence of 0.5% octyl glucopyranoside, the major cleavage products were unglycosylated alpha 1 PI and alpha 1 PI carrying one carbohydrate side chain. In contrast to the complex-type alpha 1 PI, the high-mannose type can be totally deglycosylated by endo F even in the absence of detergents. The susceptibility of the hybrid-type alpha 1 PI to endo F is between that of the complex and the high-mannose types.     

Nonselective utilization of the endomannosidase pathway for processing glycoproteins by human hepatoma (HepG2) cells.

Endo-alpha-D-mannosidase, a Golgi-situated processing enzyme, provides a glucosidase-independent pathway for the formation of complex N-linked oligosaccharides of glycoproteins (Moore, S. E. H., and Spiro, R. G. (1990) J. Biol. Chem. 265, 13104-13112). The present report demonstrates that at least five distinct glycoproteins secreted by HepG2 cells (alpha 1-antitrypsin, transferrin, alpha 1-acid glycoprotein, alpha 1-antichymotrypsin, and alpha-fetoprotein) as well as cell surface components can effectively utilize this alternate processing route. During a castanospermine (CST)-imposed glucosidase blockade, these glycoproteins apparently were produced with their usual complement of complex carbohydrate units, and upon addition of the mannosidase I inhibitor, 1-deoxymannojirimycin (DMJ), to prevent further processing of deglucosylated N-linked oligosaccharides, Man6-8GlcNAc, but not Man9GlcNAc, were identified; the Man8GlcNAc component occurred as the characteristic isomer generated by endomannosidase cleavage. Although the endomannosidase-mediated deglucosylation pathway appeared to be nonselective, a differential inhibitory effect on the secretion of the various glycoproteins was noted in the presence of CST which was directly related to the number of their N-linked oligosaccharides, ranging from minimal in alpha-fetoprotein to substantial (approximately 65%) in alpha 1-acid glycoprotein. Addition of DMJ to CST-incubated cells did not further decrease secretion of the glycoproteins, although processing was now arrested at the polymannose stage, and a portion of the oligosaccharides were still in the glucosylated form. These latter findings indicate that complex carbohydrate units are not required for secretion of these glycoproteins and that any effect which glucose residues exert on their intracellular transit would be related to movement from the endoplasmic reticulum to the Golgi compartment.     

Effect of tunicamycin and endoglycosidase H and F on the estrogen regulated 52000-Mr protein secreted by breast cancer cells

The glycosylation and immunoreactivity of an estrogen regulated glycoprotein secreted by breast cancer cells in culture and defined by its molecular mass (52 000-Mr protein) have been studied indirectly using an inhibitor of glycosylation and specific endoglycosidases. The protein and its deglycosylated forms were immunoprecipitated with specific monoclonal antibodies to the 52 000-Mr protein and analyzed by SDS polyacrylamide gel electrophoresis. The 52 000-Mr protein was intensely labelled by [3H] mannose or [35S] methionine. Tunicamycin treatment of the cells, endoglycosidase H or endoglycosidase F digestion of conditioned media, gave two identical deglycosylated forms of 50 000-Mr and 48 000-Mr which remained immunoreactive. The 48 000-Mr protein, in contrast to the 52 000 and 50 000-Mr proteins, was unable to bind concanavalin A. The 52 000-Mr protein was resolved into five spots of decreasing pI on two-dimensional gels following immunoprecipitation. Endoglycosidase H treatment decreased the molecular weight and reduced the intensity of spots of lower pI, suggesting that the N-glycosylated chains contain acidic molecules. We conclude that: The 52 000-Mr secreted protein contains at least two high mannose or hybrid N-glycosylated chains of approximately 2,000 molecular weight corresponding to 8% of the mass of the 52 000-Mr protein. The two types of monoclonal antibodies (site 1 and 2) raised against the 52 000-Mr glycoprotein are still able to recognize the 48 000-Mr N-deglycosylated form indicating that they do not interact with the N-glycosylated moiety of the molecule.     

Effect of alpha-D-mannopyranosylmethyl-p-nitrophenyltriazene on hepatic degradation and processing of the N-linked oligosaccharide chains of alpha 1-acid glycoprotein

The effects of alpha-D-mannopyranosylmethyl-p-nitrophenyltriazene (alpha-ManMNT) on the degradation and biosynthesis of oligosaccharide chains on alpha 1-acid glycoprotein (AGP) were studied. Addition of the triazene to a perfused liver prevented the complete degradation of endocytosed N-acetyl[14C]glucosamine-labeled asialo-AGP and caused the accumulation of Man2GlcNAc1 fragments in the lysosome-enriched fraction of the liver homogenate. This compound also reduced the reincorporation of lysosomally derived [14C]GlcNAc into newly secreted glycoproteins. Cultured hepatocytes treated with the inhibitor synthesized and secreted fully glycosylated AGP. However, the N-linked oligosaccharide chains on AGP secreted by the alpha-ManMNT-treated hepatocytes remained sensitive to digestion with endoglycosidase H, were resistant to neuraminidase, and consisted of Man9-7GlcNAc2 structures as analyzed by high resolution Bio-Gel P-4 chromatography. As measured by their resistance to cleavage by endoglycosidase H, the normal processing of all six carbohydrate chains on AGP to the complex form did not completely resume until nearly 24 h after triazene treatment. Since alpha-ManMNT is likely to irreversibly inactivate alpha-D-mannosidases, the return of normal AGP secretory forms after 24 h probably resulted from synthesis of new processing enzymes.