Activation- and phorbol ester-stimulated phosphorylation of a plasma membrane glycoprotein antigen expressed on mouse IL-3-dependent mast cells and serosal mast cells

As assessed by immunoprecipitation analyses, expression of the epitope recognized by the rat mAb B23.1 is approximately sevenfold greater on the surface of mouse IL-3-dependent bone marrow culture-derived mast cells (BMMC) than on serosal mast cells (SMC) obtained directly from the peritoneal cavity. Immunoprecipitation of B23.1 antibody-binding molecules from Na[125I] surface-labeled BMMC and SMC followed by sizing on SDS-polyacrylamide gels under reducing conditions demonstrated that the epitope is located on molecules of 49,000 and 47,500 Mr, respectively. An additional immunoprecipitated molecule of 42,000 Mr was detected from BMMC intrinsically radiolabeled with [35S]methionine, and pulse-chase analyses revealed that this species was a biosynthetic precursor of the 49,000 Mr cell surface form of the Ag. Treatment of the immunoprecipitated 42,000 and 49,000 Mr forms with endoglycosidase F reduced the Mr of both to 37,000, as did intrinsic radiolabeling of BMMC in the presence of tunicamycin, indicating that both the 42,000 Mr precursor form and the 49,000 Mr cell surface molecule (gp49) contained N-linked carbohydrate. Activation of [32P]orthophosphate-labeled BMMC by sensitization with mouse monoclonal IgE anti-TNP and challenge with TNP-BSA or by exposure to the calcium ionophore A23187 elicited the rapid phosphorylation of gp49 but not of its precursor forms, as did treatment of the cells with PMA. Elution of phosphorylated and immunoprecipitated gp49 from SDS-polyacrylamide gels followed by partial acid hydrolysis of the protein and phosphoamino acid analysis by high voltage thin-layer electrophoresis on cellulose plates indicated that serine, but not threonine or tyrosine, was phosphorylated upon stimulation of BMMC with IgE/Ag, calcium ionophore, or PMA. Cholera toxin did not elicit phosphorylation of gp49. These data suggest that gp49, a plasma membrane glycoprotein preferentially expressed by mouse BMMC, may be either directly or indirectly phosphorylated via protein kinase C during mast cell activation-secretion.     

The elastin associated glycoprotein gp115. Synthesis and secretion by chick cells in culture

Synthesis of gp115 by aorta smooth muscle cells and tendon fibroblasts isolated from chick embryos was investigated. gp115 was specifically immunoprecipitated by both polyclonal and monoclonal antibodies from cell lysates and culture medium of matrix free cells metabolically labeled with [3H]leucine and [35S]methionine. The component of gp115 isolated from the cell lysate had an apparent Mr in reduced sodium dodecyl sulfate polyacrylamide gels lower (105,000) than the protein isolated from the culture medium (Mr = 115,000). In immunoblot experiments, the latter corresponded in apparent Mr to the form isolated from chick tissues. gp115 was glycosylated in vitro; it was labeled with [3H]fucose, and when cells were cultured and labeled in the presence of tunicamycin, a lower Mr form with an apparent Mr = 90,000 was immunoprecipitated in both the cell lysate and the culture medium. In pulse-chase experiments, the intracellular and the extracellular forms were clearly suggestive of a direct precursor-product relationship in the absence of intermediate forms. The kinetics of secretion appeared very slow compared with that of other proteins of the extracellular matrix investigated in the same system; about 50-70% of gp115 in the form of the Mr = 105,000 species was still cell-associated after 4 h, whereas the half-time for secretion of fibronectin, type VI collagen, and tropoelastin was about 60 min, 3 h, and 60 min, respectively. Newly synthesized and processed cell-associated gp115 migrated in both reduced and non-reduced gels as a monomer. On the contrary, the secreted protein was present in the culture medium as large aggregates that did not enter the gel in the absence of reducing agents.     

Transformation of NIH 3T3 cells by DNA of the MCF-7 human mammary carcinoma cell line induces expression of an endogenous murine leukemia provirus.

Previous studies identified two glycoproteins of 86 (gp86) and 72 (gp72) kilodaltons and two nonglycosylated proteins of 70 (p70) and 19 (p19) kilodaltons which were specifically expressed in NIH cells transformed by DNA of the MCF-7 human mammary carcinoma cell line. Pulse-chase experiments and the use of tunicamycin to inhibit glycosylation suggested that gp86, gp72, and p19 were related as precursor products. Characteristics of the four transformation-associated proteins resembled those of murine leukemia virus (MuLV) proteins. Sera raised against disrupted MuLV immunoprecipitated the same four proteins in extracts of NIH(MCF-7) cells and MuLV-infected NIH 3T3 cells. In addition, a monoclonal antibody against MuLV gp70 immunoprecipitated proteins gp86 and gp72, whereas a monoclonal antibody against MuLV p15(E) immunoprecipitated gp86 and p19. These results indicate that proteins gp86, gp72, and p19 expressed in NIH(MCF-7) transformants correspond to MuLV envelope proteins gp80env, gp70, and p15(E), respectively. The transformation-associated protein p70 appears to be a non-envelope MuLV protein, most likely p65gag. Northern blot analysis confirmed that transformation of NIH cells by MCF-7 mammary carcinoma DNA led to the induction of an endogenous MuLV provirus.     

Structural analysis of the varicella-zoster virus gp98-gp62 complex: posttranslational addition of N-linked and O-linked oligosaccharide moieties.

Varicella-zoster virus specifies the formation of several glycoproteins, including the preponderant gp98-gp62 glycoprotein complex in the outer membranes of virus-infected cells. These viral glycoproteins are recognized and precipitated by a previously described monoclonal antibody designated monoclone 3B3. When an immunoblot analysis was performed, only gp98 was reactive with monoclone 3B3 antibody; likewise, titration in the presence of increased concentrations of sodium dodecyl sulfate during antigen-antibody incubations caused selective precipitation of gp98 but not gp62. Further structural analyses of gp98 were performed by using the glycosidases endo-beta-N-acetylglucosaminidase H (endoglycosidase H) and neuraminidase and two inhibitors of glycosylation (tunicamycin and monensin). In addition to gp98, antibody 3B3 reacted with several intermediate products, including gp90, gp88, gp81, and a nonglycosylated polypeptide, p73. Since gp98 was completely resistant to digestion with endoglycosidase H, it contained only complex carbohydrate moieties; conversely, gp81 contained mainly high-mannose residues. Polypeptide p73 was immunodetected in the presence of tunicamycin and designated as a nascent recipient of N-linked sugars, whereas gp88 was considered to contain O-linked oligosaccharides because its synthesis was not affected by tunicamycin. The ionophore monensin inhibited production of mature gp98, but other intermediate forms, including gp90, were detected. Since the latter product was similar in molecular weight to the desialated form of gp98, one effect of monensin treatment of varicella-zoster virus-infected cells was to block the addition of N-acetylneuraminic acid. Monensin also blocked insertion of gp98 into the plasma membrane and, as determined by electron microscopy, inhibited envelopment of the nucleocapsid and its transport within the cytoplasm. On the basis of this study, we reached the following conclusions: the primary antibody 3B3-binding epitope is located on gp98, gp98 is a mature product of viral glycoprotein processing, gp98 contains both N-linked and O-linked oligosaccharide side chains, gp90 is the desialated penultimate form of gp98, gp88 is an O-linked intermediate of gp98, gp81 is the high-mannose intermediate of gp98, and p73 is the unglycosylated precursor of gp98.     

Processing of glycoprotein gB related to neutralization of Marek's disease virus and herpesvirus of turkeys

The glycoprotein gB related to neutralization of Marek's disease virus (MDV) and herpesvirus of turkeys (HVT) is composed of several glycosylated polypeptides, which were immunoprecipitated with monoclonal antibodies and rabbit antiserum cross-reactive to MDV-gB and HVT-gB, and analyzed by SDS-polyacrylamide gel electrophoresis. The present pulse-chase experiments showed that the precursor forms of MDV- and HVT-gB were glycoproteins with molecular weights of 110K to 115K (gp115/110) and 115K (gp115), respectively. These precursor forms were processed to smaller gB's (gp63 and gp50 for MDV; gp62, gp52, and gp48 for HVT), at least in part by sialylation. The proteins synthesized in the presence of tunicamycin were two polypeptides of 88K and 83K in MDV-infected cells and a 90K polypeptide in HVT-infected cells, indicating the presence of unglycosylated precursor forms of MDV- and HVT-gB. Differences between virulent and avirulent MDV's and between HVT's with and without protective activity against Marek's disease were observed in the processed forms of MDV- and HVT-gB, especially at the processing step of sialylation.     

Biosynthesis of the epidermal growth factor receptor in A431 cells.

A monoclonal antibody R1 against the human epidermal growth factor receptor has been used to study biosynthesis in the carcinoma cell line A431. Two glycoproteins of apparent mol. wts. 95 000 and 160 000 were immunoprecipitated from cells labelled for short times with [35S]methionine or [3H]mannose. Pulse-chase studies show the 160 000 mol. wt. glycoprotein to be a precursor of the 175 000 mol. wt. receptor, but do not establish a precursor role for the 95 000 mol. wt. glycoprotein. Limited proteolysis, peptide mapping, endoglycosidase digestion and the use of monensin and tunicamycin show that the 95 000 mol. wt. glycoprotein is structurally related to the 160 000 mol. wt. glycoprotein and that both glycoproteins have approximately 22 000 - 28 000 mol. wt. of oligosaccharide side chains. Monensin blocks conversion of the 160 000 to the 175 000 mol. wt. mature receptor, a process which involves complexing several of its N-linked oligosaccharide chains. Pulse-chase studies showed that an immunoprecipitable polypeptide of 115 000 mol. wt., or 95 000 mol. wt., in the presence of monensin, was secreted into the medium at late chase times. The possible mechanisms for the origins of all the receptor-related polypeptides are discussed.     

Glucosamine hydrochloride specifically inhibits COX-2 by preventing COX-2 N-glycosylation and by increasing COX-2 protein turnover in a proteasome-dependent manner

COX-2 and its products, including prostaglandin E(2), are involved in many inflammatory processes. Glucosamine (GS) is an amino monosaccharide and has been widely used for alternative regimen of (osteo) arthritis. However, the mechanism of action of GS on COX-2 expression remains unclear. Here we describe a new action mechanism of glucosamine hydrochloride (GS-HCl) to tackle endogenous and agonist-driven COX-2 at protein level. GS-HCl (but not GS sulfate, N-acetyl GS, or galactosamine HCl) resulted in a shift in the molecular mass of COX-2 from 72-74 to 66-70 kDa and concomitant inhibition of prostaglandin E(2) production in a concentration-dependent manner in interleukin (IL)-1beta-treated A549 human lung epithelial cells. Remarkably, GS-HCl-mediated decrease in COX-2 molecular mass was associated with inhibition of COX-2 N-glycosylation during translation, as assessed by the effect of tunicamycin, the protein N-glycosylation inhibitor, or of cycloheximide, the translation inhibitor, on COX-2 modification. Specifically, the effect of low concentration of GS-HCl (1 mM) or of tunicamycin (0.1 microg/ml) to produce the aglycosylated COX-2 was rescued by the proteasomal inhibitor MG132 but not by the lysosomal or caspase inhibitors. However, the proteasomal inhibitors did not show an effect at 5 mM GS-HCl, which produced the aglycosylated or completely deglycosylated form of COX-2. Notably, GS-HCl (5 mM) also facilitated degradation of the higher molecular species of COX-2 in IL-1beta-treated A549 cells that was retarded by MG132. GS-HCl (5 mM) was also able to decrease the molecular mass of endogenous and IL-1beta- or tumor necrosis factor-alpha-driven COX-2 in different human cell lines, including Hep2 (bronchial) and H292 (laryngeal). However, GS-HCl did not affect COX-1 protein expression. These results demonstrate for the first time that GS-HCl inhibits COX-2 activity by preventing COX-2 co-translational N-glycosylation and by facilitating COX-2 protein turnover during translation in a proteasome-dependent manner.     

Identification of a putative receptor for subgroup A feline leukemia virus on feline T cells.

Retrovirus infection is initiated by the binding of virus envelope glycoprotein to a receptor molecule present on cell membranes. To characterize a receptor for feline leukemia virus (FeLV), we extensively purified the viral envelope glycoprotein, gp70, from culture supernatants of FeLV-61E (subgroup A)-infected cells by immunoaffinity chromatography. Binding of purified 125I-labeled gp70 to the feline T-cell line 3201 was specific and saturable, and Scatchard analysis revealed a single class of receptor binding sites with an average number of 1.6 x 10(5) receptors per cell and an apparent affinity constant (Ka) of 1.15 x 10(9) M-1. Cross-linking experiments identified a putative gp70-receptor complex of 135 to 140 kDa. Similarly, coprecipitation of 125I-labeled cell surface proteins with purified gp70 and a neutralizing but noninterfering anti-gp70 monoclonal antibody revealed a single cell surface protein of approximately 70 kDa. These results indicate that FeLV-A binds to feline T cells via a 70-kDa cell surface protein, its presumptive receptor.     

Expression of the Marek''s disease virus homolog of herpes simplex virus glycoprotein B in Escherichia coli and its identification as B antigen.

Marek's disease (MD) is an oncogenic disease of chickens caused by MD virus (MDV). Among the major glycoproteins found in MDV-infected cells are gp100, gp60, and gp49, detected by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis with antisera previously shown to be reactive with B antigen in immunodiffusion analysis. Following treatment with tunicamycin (TM), an inhibitor of N-linked glycosylation, the same sera were reported to detect two molecules called pr88 and pr44. However, the gene encoding B antigen was not unequivocally identified. Recently, an MDV homolog of the gene encoding herpes simplex virus glycoprotein B (gB) was identified and sequenced (L. J. N. Ross, M. Sanderson, S. D. Scott, M. M. Binns, T. Doel, and B. Milne, J. Gen. Virol. 70:1789-1804, 1989). To determine whether the MDV gB homolog gene might encode the B antigen, antisera against trpE fusion proteins of the MDV gB homolog (trpE-MDV-gB) were prepared. These antisera immunoprecipitated gp100, gp60, gp49, and a 92-kDa precursor polypeptide (pr88, now designated 92-kDa pr88, in the presence of TM) from MDV-infected cell lysates. On the basis of size comparison, trpE-MDV-gB competition and blocking assays, and the fact that gp100, gp60, gp49, and 92-kDa pr88 could be detected in MDV-infected cells with antisera specific to both MDV B antigen and the gB homolog, it was concluded that (i) the MDV gB homolog gene encodes MDV B antigen and (ii) 92-kDa pr88 is the primary precursor polypeptide. The antisera against trpE-MDV-gB also contained antibody reactive with the herpesvirus of turkey gB homolog, consistent with the known antigenic relatedness between the MDV and herpesvirus of turkey B antigens. TM inhibition data and results from pulse-chase analysis with MDV-infected cells show that MDV gB homolog processing involves cotranslational glycosylation of 92-kDa pr88 to form gp100, which is then cleaved to form gp60 and gp49, the N- and C-terminal halves, respectively, of gp100. This processing pathway is consistent with those of other gB homologs, further supporting the gene identification described above. The conclusions of this study will facilitate future research on the immunobiology of MD, especially studies on the mechanism of immunoprotection.     

In vitro translation and processing of human hepatoma cell (Hep G2) gamma-glutamyl transpeptidase

Human hepatoma cell (Hep G2) gamma-glutamyl transpeptidase (gamma-GT), a 120 ka single-chain glycoprotein, is much larger than the expected precursor of the dimeric enzyme in other human tissues. However, the Hep G2 gamma-GT mRNA encodes a 63 kDa peptide, similar to that of rat gamma-GT mRNA product and to the predicted, unglycosylated precursor of the enzyme in human tissues. Translation in presence of dog pancreas microsomes results in processing of the 63 kDa to an 80 kDa core-glycosylated species which is subsequently cleaved to 58 and 22 kDa subunits resembling those in other human tissues. The unusually large Mr of gamma-GT in Hep G2 would thus seem to be due to further glycosylation and processing in the Golgi. A deficiency of the processing protease is the most likely reason for the persistence of the single-chain form of gamma-GT in Hep G2 cells.     

A phenotypic host range alteration determines RD114 virus restriction in feline embryonic cells.

We have characterized the restriction mechanism for RD114 virus replication in embryonic feline cells (FeF). By comparing growth properties of the virus in FeF cells with its behavior in a fetal feline glial cell line (G355) permissive for RD114, we showed that both cell lines were readily infectible by virus grown in permissive cells and that no significant differences in viral integration or viral RNA expression could be detected. However, analysis of viral protein expression revealed differences in viral env gene processing in the two cell types. Envelope precursor pR85 was produced, but the expected processed gp70 product was detectable only in permissive (G355) cells. An envelope product of 85 kDa was packaged into virions produced by FeF cells, while virions produced by G355 cells contained the expected RD114 gp70. While the gp85 env-containing virions were infectious for permissive G355 cells, they were unable to infect FeF cells. The block to infection by the gp85-containing particles in FeF cells could be abrogated by treatment with the glycosylation inhibitor tunicamycin. Our results indicate that restriction of RD114 virus involves a novel mechanism dependent on two factors: altered glycosylation of the envelope to a gp85 form and an altered RD114 receptor in FeF cells.     

Tunicamycin preserves intercellular junctions, cytoarchitecture, and cell-substratum interactions in ATP-depleted epithelial cells

Pretreatment with the nucleoside antibiotic tunicamycin was found to protect cultured renal epithelial cells in the face of ATP-depletion, in large part by preserving junctional and cellular architecture. Tunicamycin pretreatment of Madin-Darby canine kidney cells not only preserved E-cadherin staining at the plasma membrane, but also inhibited ATP-depletion-mediated E-cadherin degradation. Electron microscopic analysis, together with the preservation of the staining patterns of the tight junction marker ZO-1, the apical/microvillar marker gp135, and basolateral marker Na/K-ATPase suggested that tunicamycin preserved the junctional complex and the polarized epithelial cell phenotype. Tunicamycin pretreatment also prevented reductions in the filamentous actin content of the cells, as well as preserving Golgi architecture. Moreover, a quantitative measure of cell adhesion demonstrated that tunicamycin pretreatment resulted in a fivefold increase in attachment of cells to the substratum (77% versus 16%). Thus, pretreatment with tunicamycin protects polarized epithelial cells from ischemic injury through the preservation of epithelial cell architecture, intercellular junctions, and cell-substratum interactions in the setting of intracellular ATP-depletion.     

Inhibition of Rous sarcoma virus replication by 2-deoxyglucose and tunicamycin: identification of an unglycosylated env gene product.

Two inhibitors of glycosylation, 2-deoxyglucose and tunicamycin, depressed the synthesis of infectious Rous sarcoma virus greater than 100-fold. Under the same conditions only a two- to threefold decrease in the production of virus particles was observed. The noninfectious particles had a lower density (1.145 g/ml) in isopycnic sucrose gradients and lacked the two virion glycoproteins, gp85 and gp37, found on infectious virions. The four internal structural proteins of the virus, p27, p19, p15, and p12, appeared to be assembled normally into the noninfectious virus. Polypeptides related to the Rous sarcoma virus glycoproteins were immunoprecipitated from pulse-labeled Rous sarcoma virus (Prague strain, subgroup B)-transformed cells. pr95gp, the polyprotein precursor to gp85 and gp37, was the major protein precipitated from untreated cells. PR95GP, THE POLYPROTEIN PRECURSOR TO GP85 AND GP37, WAS THE MAJOR PROTEIN PRECIPITATED FROM UNTREATED CELLS. This was absent in both tunicamycin- and 2-deoxyglucose-treatec ells, and a new polypeptide of molecular weight 57,000 to 58,000 was the major species precipitated. In tunicamycin-treated cells this product was unstable and was degraded during a 2-h chase; in 2-deoxyglucose-treated cells, on the other hand, the polypeptide appeared to be more stable and underwent partial glycosylation. The synthesis and processing of pr76, the polyprotein precursor to the internal structural proteins of the virion, occurred normally in both treated and untreated cells. It is concluded that the unglycosylated env gene product is a polypeptide of molecular weight 57,000 to 58,000.     

Influence of the N-linked oligosaccharides on the biosynthesis, intracellular routing, and function of the human asialoglycoprotein receptor

The human asialoglycoprotein receptor (ASGP-R) is a membrane glycoprotein of 46,000 Da which possesses two N-linked oligosaccharide chains (Schwartz, A. L., and Rup, D. (1983) J. Biol. Chem. 258, 11249-11255). In order to examine the role of N-linked oligosaccharides in the biosynthesis, intracellular routing, and function of the ASGP-R, we have used Hep G2 cells, which have a large number of ASGP-R, and two inhibitors of glycosylation, swainsonine and tunicamycin. In the presence of swainsonine, newly synthesized ASGP-R is a 43,000-Da species which is endoglycosidase H-sensitive, appears on the Hep G2 cell surface, and specifically binds 125I-asialoorosomucoid (ASOR). In the presence of tunicamycin newly synthesized ASGP-R is a 34,000-Da nonglycosylated species which appears on the Hep G2 cell surface where it specifically binds 125I-ASOR. There is no major effect on subsequent uptake and degradation of 125I-ASOR in cells whose ASGP-R was synthesized in the presence of tunicamycin. The turnover of ASGP-R synthesized in the presence of either swainsonine or tunicamycin is not significantly altered from that found for the normal 46,000-Da species. Thus, it appears that the two N-linked oligosaccharide chains of the human ASGP-R do not play a major role in the intracellular routing, turnover, or function of ASGP-R.     

Analysis of precursors to the envelope glycoproteins of avian RNA tumor viruses in chicken and quail cells.

Immune precipitation with monospecific antiserum was employed to study the intracellular synthesis of viral glycoproteins gp85 and gp37. Labeled gp85 and gp37 were detected from lysates of cells transformed with Rous sacroma virus, strain B77, after long-term labeling with radioactive glucosamine or phenylalanine. Immune precipitates prepared from lysates of cells pulse-labeled for a short time resulted in a glycoprotein of 92,000 molecular weight (gp92). This precursor was stable in B77-transformed Japanese quail cells for several hours, whereas in chicken cells it could be chased within a few hours into virion glycoproteins gp85 and gp37. Similarly, the precursor for the structural viral proteins, pr76, persisted in quail cells much longer than in chicken cells. During very short pulses or in the presence of a glucosamine block (25 mM glucosamine), the antiserum against the viral envelope glycoproteins detected a precursor of higher electrophoretic mobility of approximately 70,000 molecular weight, "p70." Fucose label entered gp92 and gp85 as well as "p70." Proteolytic treatment of virion-bound gp85 in vitro generated two discrete glycoproteins of 62,000 and 45,000 molecular weight, but did not result in an increase in the amount of gp37.     

Characterization of stable Chinese hamster ovary cells expressing wild-type, secreted, and glycosylphosphatidylinositol-anchored human immunodeficiency virus type 1 envelope glycoprotein.

We generated Chinese hamster ovary cell lines that stably express wild-type, secreted, and glycosylphosphatidylinositol (GPI)-anchored envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1). The cells expressing wild-type Env (WT cells) express both the precursor gp160 and the mature gp120/gp41 and readily form large syncytia when cocultivated with CD4+ human cells. The cells expressing secreted Env (SEC cells) release 140-kDa precursor and mature 120-kDa envelope glycoproteins into the supernatants. The cells expressing GPI-anchored Env (PI cells) express both 140-kDa precursor and mature gp120/gp41 envelope glycoproteins, which can be released from the cell surface by treatment with phosphatidylinositol-specific phospholipase C (PI-PLC). Both the secreted and PI-PLC-released envelope glycoproteins form oligomers that can be detected on nonreducing sodium dodecyl sulfate-polyacrylamide gels. In contrast to the WT cells, the SEC and PI cells do not form syncytia when cocultivated with CD4+ human cells. The availability of cells producing water-soluble oligomers of HIV-1 Env should facilitate studies of envelope glycoprotein structure and function. The WT cells, which readily induce syncytia with CD4+ cells, provide a convenient system for assessing potential fusion inhibitors and for studying the fusion mechanism of the HIV Env glycoprotein.     

Synthesis, processing, and secretion of the Marek''s disease herpesvirus A antigen glycoprotein.

The 57,000- to 65,000-dalton (Da) Marek's disease herpesvirus A (MDHV-A) antigen glycoprotein (gp57-65) has a 47,000-Da unglycosylated precursor polypeptide (pr47), as determined by immunological detection after cell-free translation of infected-cell mRNA. Cleavage of its signal peptide yielded a 44,000-Da precursor polypeptide molecule (pr44), detected both in vivo after tunicamycin inhibition of glycosylation and in vitro after dog pancreas microsome processing of pr47. High-resolution pulse-chase studies showed that pr44 was quickly glycosylated (within 1 min) to nearly full size, a rapid processing time consistent with a cotranslational mode of glycosylation. This major glycosylation intermediate was further modified 6 to 30 min postsynthesis (including the addition of sialic acid), and mature MDHV-A was secreted 30 to 120 min postsynthesis. Limited apparent secretion of pr44 occurred only in the first minute postsynthesis, in contrast to the later secretion of most of the MDHV-A polypeptide as the fully glycosylated form described above. In addition, in the presence of tunicamycin a small fraction of the newly synthesized MDHV-A protein appeared as a secreted, partially glycosylated, heterogeneously sized precursor larger than pr44. pr44 constituted the major fraction of the new MDHV-A made in the presence of the inhibitor but the precursor was smaller than mature MDHV-A. These data indicate that there is a minor glycosylation pathway not sensitive to tunicamycin and that "normal" glycosylation is not necessary for secretion. Collectively, the data demonstrate that the rapid release of most of the fully glycosylated form of MHDV-A from the cell shortly after synthesis is true secretion in a well-regulated and precisely programmed way and not the result of cell death and disruption.